Project Summary Colorectal cancer (CRC) is predicted to be the second leading cause of cancer-related deaths in 2018. Greater than 90% of CRC tumors contain a mutation in the canonical Wnt-signaling pathway, which deregulates ?-CATENIN-TCF transcriptional activity and drives oncogenic gene expression. Indeed, the canonical Wnt-target LGR5 is proposed mark CRC stem cells, which are required for primary and metastatic tumor growth. In addition to genetic mutations, CRC tumors bear alterations in epigenetic features, including global DNA hypomethylation, CpG island hypermethylation and a variety of changes in histone post-translational modifications. Although these chromatin changes are thought to be important for CRC tumorigenesis, the oncogenic chromatin factors in CRC remain largely undefined, impeding targeting of epigenetic processes in the clinic. To address this gap in knowledge, I conducted an in vitro CRISPR-Cas9 dropout screen using a chromatin-focused pooled lentiviral guide RNA (gRNA) library in a primary murine CRC model. gRNAs targeting Kmt5a (encoding SETD8), the sole histone 4 lysine 20 (H4K20) monomethyltransferase, dropped out significantly during this screen, indicating the importance of this gene in CRC proliferation. SETD8 and H4K20me1 play important roles in regulating transcription, DNA damage repair and cell cycle progression. Particularly relevant for CRC, SETD8 methyltransferase activity is required for ?-CATENIN-TCF transcriptional function in human cell lines, zebrafish and Drosophila. Indeed, upon Wnt stimulation, SETD8 directly interacts with ?-CATENIN-TCF complexes and deposits H4K20 monomethyl (H4K20me1) modifications at canonical Wnt target genes. Based on these findings, I hypothesize that SETD8 promotes CRC tumorigenesis in part by cooperating with ?-CATENIN-TCF to potentiate the canonical Wnt-signaling pathway. To this end, in Aim 1 I will investigate the importance of SETD8 in CRC tumorigenesis in vivo by manipulating SETD8 expression in murine CRC organoids and orthotopically implanting these lines into a syngeneic host. Moreover, I will define an in vitro therapeutic index for the SETD8 inhibitor UNC-0379 in pairs of primary normal and CRC organoids derived from human patients. In Aim 2 I will elucidate the mechanistic role of SETD8 in ?-CATENIN-TCF transcriptional activity by manipulating SETD8 levels and measuring canonical Wnt-target activation by transcriptome profiling and functional reporter assays, as well as by quantifying the abundance and activity of Lgr5+ CRC stem cells in vivo. I will also define the protein-protein interactions between ?-CATENIN, the TCF factor LEF1 and SETD8 as well as the enrichment of these proteins and H4K20me1 at canonical Wnt-target loci. This study will determine, for the first time, the role of SETD8 in CRC tumorigenesis. The proposed experiments will potentially identify a promising therapeutic avenue to combat this deadly disease.